Although the p53 signaling pathway is perhaps one of the best studied pathways in the human cell, there are many open questions related to its regulation and function. We understand a great deal about specific protein- protein interactions in the pathway, but we understand little about the overall relationship between input (damage and stress signals) and output (cellular outcomes ranging from cell growth to cell death), and the kinetics of the response that determines these relationships. The supplementary funding I request here would allow my lab to take two novel approaches to these questions. In Aim 1, we will follow up on a recent discovery, made using long-term single-cell imaging, that p53 shows spontaneous pulses in the absence of external DNA damage. These pulses appear to be connected to the cell cycle and we therefore suspect that they reflect spontaneous damage during DNA replication or mitosis. We will ask what triggers p53 spontaneous pulses and what controls their shape and timing. We will use a system we have developed for quantifying the amount of double-stranded DNA breaks in live cells to determine whether p53 spontaneous pulses correlate with spontaneous DNA damage during cellular growth and division. We will use chemical and genetic perturbation to test which regulators initiate and control p53 spontaneous pulses. Understanding the regulation of p53 spontaneous pulses will give us the tools to manipulate them, and to develop approaches to understand their function. It is possible that these spontaneous p53 pulses have an important role in preventing the build-up of DNA damage in unstressed cells - a previously unappreciated feature of p53's activity. We have preliminary data showing that p53 pulses in non-stressed conditions do not activate p21 and only a subset of the cells activates p21 in response to irradiation. We suspect that this is due to differences in the post-translational modifications on p53 in these cells. In Aim 2 we will use chemical and genetic perturbations to force, or prevent specific p53 modifications and test the effect on p21 dynamics and on cell fate using live cell imaging. We will also determine the post-translational modification of p53 during the first and second pulse and will use flow cytometry to sort and isolate cells that activate p21 versus cells that do not. We will then use mass spectrometry to determine which p53 modifications occur in these different populations. This will allow us, for the first time, to take an unbiased approach for connecting combinations of p53 modifications with the activation of its specific target genes. PUBLIC HEALTH RELEVANCE: Our study will provide new insights into the control and manipulation of the p53 pathway, perhaps the most important pathway protecting human cells against the development of cancer. These studies will give a deeper understanding of the biological mechanisms and function of p53, and will provide a prototype for the analysis, description, and understanding of the dynamics of other signaling pathways in single living human cells.